The plasminogen activators, tissue plasminogen activator (t-PA) and urokinase (u-PA), consist mainly of two parts, a catalytic part which is involved in the conversion of plasminogen to plasmin (a serine protease which degrades the fibrin network of a blood clot) and a non-catalytic part which contains the regulatory regions responsible for physiological specificity such as fibrin binding. Van Zonneveld et al., Proc. Nat'l. Acad. Sci. U.S.A., 83 4670-4674 (1986) has elucidated the functions of several structural domains of t-PA.
The non-catalytic part of t-PA contains at least three domain structures: two kringles of 82 amino acids each; an epidermal growth factor (EGF) region of 45 amino acids and a fibronection (finger) domain of 45 amino acids. Urokinase has a similar non-catalytic domain structure with a single kringle of 82 amino acids and no fibronection region.
The EGF domains of t-PA and u-PA share considerable regions of homology with the human epidermal growth factor sequence. Vehar et al., Bio/Technology, 1051-1057, December 1984 depicts these homologous regions in FIG. 7, page 1055. For example, the relative positions of five of the six cysteine residues, involved in disulfide bonding, are conserved. Similarly, several amino acids and amino acid sequences within the EGF domain of t-PA and u-PA correspond to the same positions in EGF; e.g. amino acids 52, 56-62, 67-69, 73-76, 79, 81 and 84-87.
Because of its high binding specificity for blood clot fibrin, t-PA is regarded as an ideal thrombolytic agent [Collen, Circulation, 72 1820 (1985)]. However, t-PA is very rapidly removed from the circulation by the liver (t.sub.1/2 =about 1 to 3 minutes), thereby reducing its effectiveness in the treatment of thrombotic vascular accidents [Emeis et al., Thrombosis and Haemostasis, 54 (3), 661-664 (1985)]. Fuchs et al., Blood, 65 (3) 539-544 (1985) demonstrated that urokinase (u-PA) is similarly removed from the circulation by the liver and that these plasminogen activators are cleared by a process not dependent upon the proteinase active site. The hepatocyte was shown to be the cell-type responsible for rapid t-PA clearance. Rijken et al., Biochem. J. 238 643-644 (1986) found that the light chain of t-PA (C-terminal portion) was much less rapidly cleared from the circulation than the heavy chain, establishing the heavy chain as containing the polypeptide sequence recognized by the liver. The binding of t-PA and urokinase to liver membranes is fast and is not inhibited by asialo-fetuin (terminal galactose), mannan or fibrinogen, suggesting that carbohydrates do not mediate its clearance from the blood.